Ligand Recognition of the Major Birch PollenAllergen Bet v 1 is Isoform DependentChristian Seutter von Loetzen1*, Thessa Jacob1, Olivia Hartl-Spiegelhauer1,Lothar Vogel3, Dirk Schiller3, Cornelia Spörlein-Güttler2, Rainer Schobert2, Stefan Vieths3,Maximilian Johannes Hartl1, Paul Rösch1
1 Department of Biopolymers, University of Bayreuth, Bayreuth, Bavaria, Germany, 2 Chair of OrganicChemistry, University of Bayreuth, Bayreuth, Bavaria, Germany, 3 Division of Allergology, Paul-Ehrlich-Institut, Langen, Hesse, Germany
AbstractEach spring millions of patients suffer from allergies when birch pollen is released into the
air. In most cases, the major pollen allergen Bet v 1 is the elicitor of the allergy symptoms.
Bet v 1 comes in a variety of isoforms that share virtually identical conformations, but their
relative concentrations are plant-specific. Glycosylated flavonoids, such as quercetin-3-O-
sophoroside, are the physiological ligands of Bet v 1, and here we found that three isoforms
differing in their allergenic potential also show an individual, highly specific binding behav-
iour for the different ligands. This specificity is driven by the sugar moieties of the ligands
rather than the flavonols. While the influence of the ligands on the allergenicity of the Bet v 1
isoforms may be limited, the isoform and ligand mixtures add up to a complex and thus indi-
vidual fingerprint of the pollen. We suggest that this mixture is not only acting as an effective
chemical sunscreen for pollen DNA, but may also play an important role in recognition pro-
cesses during pollination.
IntroductionAllergies are a major health problem worldwide. In particular, type I or immediate type aller-gies [1] that involve proteins as causative agents are very widespread and potentially severe.The major birch pollen allergen Bet v 1 from the European white birch (Betula verrucosa)alone [2] affects an estimated 100 million people [3]. Although birch pollen contain a variety ofallergens from different protein families, more than 60% of all birch pollen-allergic patientsreact exclusively to Bet v 1 [4]. Up to 90% of the Bet v 1-sensitized patients also exhibit IgE-me-diated allergic cross-reactions (oral allergy syndrome) to Bet v 1-homologous food allergens,with fruits, vegetables, and nuts as the most important elicitors of the allergy [5,6].
On the basis of sequence similarities and the protein three-dimensional structures, Bet v 1and related pollen and food allergens belong to the family of class 10 pathogenesis-related pro-teins (PR-10) within the Bet v 1 superfamily. It was suggested that proteins in this family are in-volved in plant defense mechanisms, since expression of the respective genes is induced upon
PLOSONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 1 / 20
OPEN ACCESS
Citation: Seutter von Loetzen C, Jacob T, Hartl-Spiegelhauer O, Vogel L, Schiller D, Spörlein-GüttlerC, et al. (2015) Ligand Recognition of the Major BirchPollen Allergen Bet v 1 is Isoform Dependent. PLoSONE 10(6): e0128677. doi:10.1371/journal.pone.0128677
Academic Editor: Eugene A. Permyakov, RussianAcademy of Sciences, Institute for BiologicalInstrumentation, RUSSIAN FEDERATION
attacks of pathogens and by environmental stress [7]. However, the physiological roles of PR-10 proteins seem to extend beyond stress and pathogen response. Thus, the PR-10 strawberryallergen Fra a 1 is involved in controlling flavonoid biosynthesis and this protein is capable ofbinding different metabolic intermediates [8]. In general, PR-10 proteins often co-occur withflavonoids in vivo [9–15] and interact with flavonoids in vitro [8,16], as clearly evidenced, forexample, for Bet v 1 [17,18]. Why many, if not all, PR-10 proteins appear as mixtures of iso-forms, however, remains elusive [19–21].
The first Bet v 1 isoform described on the DNA level was Bet v 1a [22] followed by the iden-tification of numerous other isoform sequences [23–25]. At least 18 Bet v 1 variants found inpollen on the mRNA or protein level [23,26,27] are officially listed as isoallergens (http://www.allergen.org). Studies on the proteomic profile of birch pollen extracts of different origin or spe-cies revealed significant differences of isoform composition and quantity [26,27]. For example,Bet v 1 constitutes up to 30% of the total protein content in Swedish pollen and 12% in Aus-trian pollen. In all cases so far, the most abundant isoform is Bet v 1a (50% to 70%), followedby Bet v 1d (20%), Bet v 1b (3% to 20%), Bet v 1f (2% to 8%), and Bet v 1j (~1%) [26].
Bet v 1a is well characterized by biochemical [2,18,28] and structural [29–31] studies. Thelarge hydrophobic pocket formed by the secondary structure elements of Bet v 1 suggested thatthis allergen acts as storage or carrier protein [29,32,33]. Previous research work focused ontrial-and-error approaches or docking simulations to test various ligands for binding to recom-binant Bet v 1 [18,30,34]. We recently purified Bet v 1 in complex with its natural ligand quer-cetin-3-O-sophoroside (Q3OS) directly from mature birch pollen and confirmed binding byreconstitution of the Bet v 1a:Q3OS complex from its recombinant protein and synthetic ligandcomponent [17]. We hypothesized that this complex may be involved in UV-protection of thepollen DNA and that Q3OS may stimulate pollen tube formation upon rehydration of the pol-len. We then asked why different isoforms exist and whether there are physiological ligandsother than Q3OS. Although it is tempting to believe on the basis of the high sequence identitiesof 87.4%–99.4% to Bet v 1a that all isoforms specifically interact with Q3OS, Bet v 1 isoformsare strikingly different in their immunological and allergenic properties [35] and, although al-lergenicity is mainly correlated with binding epitopes at the surface of allergens [36] it has al-ways been speculated that Bet v 1 proteins as such are only part of the story, and that IgEbinding needs to be tested in complex with their natural binding partners to arrive at meaning-ful results [30].
In order to characterize serological IgE binding as a measure for allergenicity as well as thephysiological function of Bet v 1, we thoroughly studied ligand- and antibody-binding behav-iour of the Bet v 1 isoforms a (hyperallergen), m (intermediate), and d (hypoallergen). Surpris-ingly, while none of the ligands significantly alters the allergenicity of Bet v 1, ligand binding tothe different isoforms is diverse and highly dependent on the composition of the ligands’sugar moieties.
Results and Discussion
Bet v 1:Q3OS interaction is isoform-dependentWe were asking whether isoforms a, d, and m form identical complexes with the Bet v 1a natu-ral ligand Q3OS [17]. In an initial experiment we noticed that Q3OS exhibits slightly differentshades of yellow when incubated with these Bet v 1 isoforms. After incubation we removed ex-cess Q3OS with a G25 column and recorded UV/VIS absorption spectra of the protein frac-tions (Fig 1A) and of unbound Q3OS (Fig 1B). In the presence of Bet v 1a, the UV/VISspectrum of Q3OS shows a clear shoulder around 360 nm, while this is not the case for Bet v 1
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 2 / 20
isoforms d or m. These absorbance differences suggest that the putative Bet v 1d:Q3OS and Betv 1m:Q3OS complexes are different from the Bet v 1a:Q3OS complex.
Binding of unglycosylated flavonoids to Bet v 1 isoformsSince the determination of the three-dimensional structure of Bet v 1a in 1996 [29] it has beensuggested that the protein functions as a carrier or storage protein. The existence of varioushighly similar, structurally almost identical isoforms could be evidence for a complex networkof different acceptors, targeted to bind chemically similar ligands. Hitherto, there is only limit-ed comparable information available about differences in ligand binding behaviour betweenBet v 1 isoforms of different allergenic potential. Recent approaches used indirect methods(ANS replacement assay, [18]) or analysed ligand binding in protein crystals [30,37]. We nowused UV/VIS and NMR spectroscopy to systematically analyse and compare binding of physio-logically relevant ligands to three different Bet v 1 isoforms (Fig 1C) in solution, with a focuson flavonoids.
Fig 1. UV/VIS spectroscopy of flavonoids and Bet v 1 isoforms. All spectra were recorded at 298 K with 50 mM sodium phosphate, 50 mMNaCl at pH 7.0and 10%DMSO as sample buffer. A Binding of Q3OS to Bet v 1 isoforms. UV/VIS spectra of 20 μMBet v 1a (-) and Q3OS incubated with Bet v 1a (-), Bet v1d (-) and Bet v 1m (-) concentrated and subsequently eluted from a G25 column.B UV/VIS spectra of 20 μMQ3OS (-), quercetin (-), Q3OGlc (-) andQ3OGal (-) reveal differences in absorption maxima and intensities. C Sequence alignment of the Bet v 1 isoforms a, d and m as performed with ClustalW[91]. Amino acids are marked with asterisks (identical), colons (conserved) and dots (semi-conserved). Residues that vary compared to Bet v 1a arehighlighted in red for Bet v 1d (95.6% sequence identity to Bet v 1a) and in blue for Bet v 1m (89.3% sequence identity to Bet v 1a).
doi:10.1371/journal.pone.0128677.g001
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 3 / 20
A set of five different flavonoids was used to analyse the influence of number and positionof hydroxyl groups of the flavonoid moiety during binding to Bet v 1 isoforms (Table 1 andS1A to S1E Fig). UV/VIS and chemical shift perturbation (CSP) measurements with 1H-15NHSQC NMR spectroscopy were performed to study affinities and binding sites of various flavo-noids. The UV/VIS spectra from the titration experiment of naringenin and Bet v 1a show iso-sbestic points indicating a two-state binding process with a Kd of roughly 60 μM (Fig 2A and2B). In the 1H-15N HSQC spectra of 15N-Bet v 1a with increasing concentration of naringenin,the G140 resonance was in the intermediate exchange regime, but gradual CSPs were observedfor the majority of affected resonances (Fig 2C, S1 Table), from which a Kd value of approxi-mately 30 μM could be estimated (Fig 2D and 2E). The CSP mapping on the Bet v 1a:narin-genin structure (pdb code 4A87, [30]) agreed well with the results from X-ray crystallography(Fig 2F). We confirmed F22, Y83, I102, and E141 as interacting residues (S1 Table) with
Table 1. Dissociation constants for Bet v 1 isoform interaction with flavonoids and sugars.
Dissociation constant Kd (μM)
Flavonoid Method Bet v 1a Bet v 1m Bet v 1d
Flavone UV/VIS n.a. 1 n.a. - 2
NMR 67.1±12.1 213.3±36.6 69.9±14.8
Naringenin UV/VIS 60.6±3.2 28.1±0.8 37.7±6.4
NMR 30.0±7.0 22.1±5.5 -
Fisetin UV/VIS 14.3±1.1 68.6±12.4 13.9±2.1
NMR 37.2±6.5 85.1±21.7 -
Quercetin UV/VIS 9.2±0.6 26.5±1.5 10.2±1.0
NMR 31.4±10.3 65.8±8.2 -
Myricetin UV/VIS 4.2±0.7 n.a. 1.2±0.2
NMR 14.6±6.5 99.3±19.4 -
Glucose UV/VIS - - -
NMR No binding No binding No binding
Galactose UV/VIS - - -
NMR No binding No binding No binding
Q3OGlc UV/VIS n.a. n.a. -
NMR 288.4±24.0 <5 No binding
Docking - 0.2–6.1 -
Q3OGal UV/VIS n.a. n.a. -
NMR <5 <5 No binding
Docking 3.2–14.8 0.4–10.4 -
Q3OS UV/VIS n.a. n.a. -
NMR <1 No binding No binding
Fluorescence 0.57 [17] - -
Docking 0.1–1.7 - -
Kd values from UV/VIS titration experiments were determined by non-linear regression analysis. The error represents the standard error of the best fit
according to Eq 1. The dissociation constants determined with NMR spectroscopy represent an averaged Kd app value of all analysable residues showing
CSPs > 0.08 ppm (S1 to S3 Tables) with the corresponding standard deviation. Kd ranges from docking simulation were obtained from binding energies
for ligands docked inside the hydrophobic pocket of Bet v 1a and Bet v 1m.1 not analysable (n.a.)2 not measured (-)
doi:10.1371/journal.pone.0128677.t001
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 4 / 20
Fig 2. Binding of naringenin to Bet v 1a. A, UV/VIS spectra of the equilibrium titration of 20 μM naringenin with Bet v 1a. All spectra were recorded at 298 Kwith 50 mM sodium phosphate, 50 mM NaCl at pH 7.0 and 10%DMSO as sample buffer. B, Absorbance changes at 325 nm plotted against the Bet v 1aconcentration as shown for the data in A. The curve represents the best fit to Eq (1) resulting in a Kd value of 60.6 ± 3.2 μM. C, Overlay of six 1H-15N HSQCspectra of 100 μMBet v 1a in the presence of increasing naringenin concentrations from light to dark red. The experiments were performed with a BrukerAvance 700 MHz spectrometer in 50 M sodium phosphate, 50 mMNaCl, pH 7.0 and 10% 2H2O at 298 K. Naringenin was added from a stock prepared indeuterated DMSO to a final excess of 1:4.5 over Bet v 1a and a final DMSO concentration of 10%. D, Chemical shift changes (Δδnorm) calculated with Eq (2)for residues A15 (�) and G89 (●) plotted against the ration of naringenin:Bet v 1a during titration. The curves represent the best fit to a quadric binding equationfrom the analysis software of NMRViewJ [89] (S1 Table). E, Calculated Δδnorm values upon naringenin addition plotted against the Bet v 1a amino acidsequence and F, mapped on a cartoon representation of the complex structure of Bet v 1a:naringenin (pdb code: 4A87) with 0.04 ppm� Δδ� 0.08 ppmshown as yellow; 0.08 ppm� Δδ� 0.12 ppm shown as orange; and 0.12 ppm < Δδ shown as red. Bet v 1a in grey, naringenin in green sticks, oxygen in red.
doi:10.1371/journal.pone.0128677.g002
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 5 / 20
CSPs> 0.12 ppm and the reported change in side chain conformation of K137 [30] could alsobe observed as large CSP with a Δδnorm value of 0.27 ppm.
The Kd values of all tested flavonoids were in the medium to low micromolar range(Table 1, S1 to S3 Tables). We observed shifts of the UV/VIS absorption maxima and isosbesticpoints in the spectra upon Bet v 1 addition for all isoforms and flavonoids (S4 Table). During1H-15N HSQC titration, the majority of affected Bet v 1 resonances were in the fast exchangeregime with the highest Kd generally for the non-hydroxylated flavone. Thereby, the significantCSPs obtained during titration were generally spread over the sequence of each isoform, mak-ing it difficult to predict a precise binding site for flavone. Due to hydrophobic interactions, fla-vone seems to bind more flexibly and somewhat more weakly inside the hydrophobic pocket.
In general, we obtained the best results (lowest standard error) for our titration experimentsby fitting the data to an equation corresponding to a simple bimolecular reaction (Eq 1 and asimilar equation provided by the NMRviewJ software). Prior experiments on flavonoid bindingto other allergens of the PR-10 class performed so far also suggested a single site bindingscheme to be valid [8,30]. Therefore, it seems as there is only one binding site for flavonoids in-side the Bet v 1 hydrophobic pocket.
While the position of hydroxyl groups is insignificant, the addition of such leads to a signifi-cant decrease of Kd for flavonoids interacting with Bet v 1 isoforms a and d. Myricetin containssix hydroxyl groups and shows a 15-fold higher affinity to Bet v 1a (4.2 μM) and an even60-fold higher affinity to Bet v 1d (1.2 μM) than flavone (Table 1). Those affinities are charac-teristic for a change of resonance positions and shapes in the form in the fast exchange regimeto the intermediate exchange regime on the NMR time scale ([38], S1 Table). Accordingly, inthe presence of myricetin, almost half of the affected resonances (11 of 28 residues) of Bet v 1aare in the intermediate exchange regime (S1 Table). Bet v 1m generally shows lower affinitiestowards the tested flavonoids compared to Bet v 1 isoforms a and d. Furthermore, the Kd valuesseem to be independent of the number of flavonoid hydroxyl groups. However, the presence ofa hydroxyl group at C5’ in the B-ring of fisetin and myricetin decreases the affinity towards Betv 1m compared to naringenin and quercetin (Table 1).
The interaction surfaces of all flavonoids are located inside the hydrophobic pocket of Bet v1 but vary between Bet v 1a (T7 to S11, I23 to N28, F64, G89 to I91, I102, K115 to N118, K137 to E141,and R145) and Bet v 1m (T57, G89 to G92, I102, K137 to L143). Most likely, flavonoids enter the hy-drophobic pocket via one of the two gaps formed by the mostly nonpolar residues F62, P63, F64,P90, Q132, A135, S136, and M139 (entrance 1) or by residues I23, L24, D25, D27, T52, K54, Y81, andI102. The third gap, Y5, T7, V133, and K137 with a diameter of ~6 Å, is probably too small for fla-vonoids to enter the cavity [30].
Despite the observed differences between the three isoforms with respect to binding ofunglycosylated flavonoids, the hydrophobic cavity of Bet v 1 isoforms seem to be promiscuousacceptors of small hydrophobic and amphiphilic molecules in vitro. However, the vast majorityof naturally occurring flavonoids are modified with additional functional groups such as meth-yl ether groups, glycosylations, or combinations of these [39]. In addition, the low water solu-bility of unglycosylated flavonoids [40] and their low potential physiological concentration inpollen [41] is not necessarily indicative of a major physiological importance ofthese complexes.
Binding of glycosylated flavonoids is governed by the sugar moietyAs no isoform-specific binding pattern for unglycosylated flavonoids could be derived, we fo-cused on the sugar moiety of the quercetin glycosides quercetin-3-O-sophoroside (Q3OS),quercetin-3-O-glucoside (Q3OGlc), and quercetin-3-O-galactoside (Q3OGal) as binding
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 6 / 20
partners of Bet v 1 isoforms (Table 1 and S1F to S1H Fig). UV/VIS absorption spectra showmaxima of different intensities at physiological pH for Q3OGlc at 364 nm, and for Q3OGaland Q3OS at 358 nm (Fig 1B), but the spectral changes on Bet v 1 binding were too small to beanalysed with confidence. Thus we resorted to 1H-15N HSQC spectroscopy for further studies.
Titration of Bet v 1a with Q3OS resulted in a change of resonance positions on the interme-diate to slow exchange limit on the NMR time-scale for 16 residues (F22, L29, I38, K55, R70, E73,V74, N82, S84, V85, K115, Y120, K137, E138, G140, and L144), with a resulting Kd of 566 ± 85 nM([17], Fig 3A and 3B, S1 Table). Although Q3OGlc is simply shortened by a single glucose moi-ety compared to Q3OS, the Bet v 1a:Q3OGlc Kd of 288 μM is three orders of magnitude higherthan that of Bet v 1a:Q3OS (Fig 3C and S2A Fig). In contrast, Bet v 1a shows high affinity toQ3OGal with resonances of 16 residues in intermediate exchange (F22, I23, G26, K54, F64, R70,E73, D93, K115, S136 to E141, and L144) and 11 residues in the fast exchange regime (T10, I53, T66,G92, L95, V128, Q132, V133, A135, T142, and V147) showing CSPs> 0.04 ppm (Fig 3D and S2B Fig,S1 Table). According to docking simulations, Q3OGal binds in the hydrophobic pocket of Betv 1a, with the sugar moiety either completely inside or at the opening of the pocket (entry ε1,[30]) at the flexible loop connecting β7 with α3. Since we observed the majority of affected res-onances in the intermediate exchange regime, we concluded that the affinity of Bet v 1a toQ3OGal is higher than for its aglycon quercetin (9.6 μM) and estimated the Kd-value< 5 μM.Affinity scores of the models resulted in Kd values from 3.2 μM to 14.8 μM (Table 1). Obvious-ly, stereochemical changes in the sugar moiety of flavonol glycosides can strongly influence theaffinity to Bet v 1a.
Although Bet v 1d binds flavonoids with affinities comparable to those of Bet v 1a (Table 1),it shows only very weak affinity for the glycosylated flavonoids that we have analysed here. Re-markably, even a 15-fold excess of Q3OS, Q3OGlc or Q3OGal (Fig 3E to 3H, S2C and S2D Fig)did not produce significant CSPs for Bet v 1d.
Furthermore, titration of Bet v 1m with Q3OS also did not lead to significant CSPs (Fig 3Iand 3J), suggesting that Bet v 1a:Q3OS formation is highly specific. However, in contrast to Betv 1a and d, Bet v 1m strongly binds to Q3OGlc (estimated Kd < 5 μM), with resonances of 21residues in intermediate exchange (E6, I23, G26, I38, T57, F64, Y66, G89 to G92, I98, and I136 toL144) and four residues (A34, E87, E96, and V147) with CSPs> 0.04 ppm (Fig 3K and S2E Fig, S2Table). The docking simulation suggested Q3OGlc to bind in the hydrophobic pocket of Bet v1m with Kd values of 0.4 μM to 10.4 μM. Bet v 1m also shows high affinity for Q3OGal with 21intermediate exchanging residues (I38, S39, T57, F64, Y66, M85 to E87, G89 to G92, E96, K134, I136
to E141, L143, and L144) and 14 residues (D25, A34, A37, V41, N47, I56, E87, G88, T94, L95, K115,T122, K123, and A135) with CSPs> 0.04 ppm (Fig 3L and S2F Fig, S2 Table) and Kd values ob-tained from docking simulations between 0.2 μM and 6.1 μM (Table 1).
Although glycosylation drastically changed the binding behaviour of quercetin to the vari-ous Bet v 1 isoforms, glucose and galactose alone showed no detectable affinity to any isoform(Table 1).
Bet v 1d varies in seven amino acids (T7I, F30V, S57N, I91V, S112C, I113V, and D125N;Fig 1C) compared to Bet v 1a. Thus, strong specific binding and virtual lack of such is achievedby variation of just seven or even fewer amino acids. None of those seven variable residues,however, is directly involved in Q3OS or Q3OGal binding in Bet v 1a or is part of the aminoacids which form the potential entrances. T7 is part of the third opening in Bet v 1a, which ispresumably too small for glycosylated flavonoids entrance. The loss of affinity might be ex-plained by a slightly different structural arrangement of Bet v 1d, which could result in varia-tions in the openings to the hydrophobic pocket. In contrast to Bet v 1d, Bet v 1m shows fourvariations in entrance 1 (F62S, P90A, Q132H, and S136I compared to Bet v 1a; Fig 1C) whichare likely to directly block the access route for Q3OS, but not for Q3OGlc and Q3OGal, into
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 7 / 20
Fig 3. Binding of quercetin glycosides to Bet v 1 isoforms. All experiments were performed with 50 μM(Q3OS) or 100 μM (Q3OGlc, Q3OGal) 15N-uniformly labelled Bet v 1 isoforms at 298 K in 50 mM sodium
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 8 / 20
the hydrophobic pocket. Substitutions of amino acids in the C-terminal helix (S136I, M139K,and T142A) could contribute to an increased affinity to Q3OGlc as compared to Bet v 1a as theC-terminal helix determines size and character of the hydrophobic cavity in PR-10 proteins[33].
In addition to structural aspects, a phenomenon known as enthalpy-entropy compensation[42] can explain the binding behaviour of the isoforms to glycosylated flavonoids and the sug-ars alone. Upon Bet v 1 isoform–ligand complexation, water molecules that form the hydrationshell of the sugar moiety and the binding cavity will tend to escape to the bulk with a concomi-tant decrease or increase in entropic energy contribution, depending on the pre-existing molec-ular interactions. This event is accompanied by the increase or decrease of degrees of freedomfor the ligand and the residues forming the binding site. The setup of solvent clusters on thesurface of the protein-ligand complex also contributes to the overall binding affinity with en-thalpy/entropy gains (Bet v 1a:Q3OS or Q3OGal; Bet v 1m:Q3OGlc or Q3OGal), penalties (Betv 1a:Q3OGlc), or even complete abolishment of observable binding (Bet v 1d:Q3OS, Q3OGlcor Q3OGal; Bet v 1m:Q3OS) compared to the aglycon quercetin. Similar effects have been re-ported and seem to be generally characteristic for each ligand/receptor involved [43–45]. In ad-dition, glucose and galactose alone showed no detectable affinity to any isoform (Table 1). Thepotential enthalpy gains upon carbohydrate interaction with proteins are often counteractedby the above described change of entropy [42], resulting in the abolishment of binding. We ob-served this effect already for the binding of sophorose to Bet v 1a [17].
In summary, our results firmly suggest that Bet v 1:ligand binding is isoform-specific andthat the binding specificity is entropically driven by the sugar moiety. Glycosylation of querce-tin can thereby significantly increase the affinity compared to the aglycon (Table 1). The hy-drophobic pockets formed by Bet v 1 isoforms are obviously designed for specificdiscrimination between the sugar moieties of glycosylated flavonoids.
Allergenicity of Bet v 1 isoforms is unaffected by ligandsBet v 1 isoforms can be grouped into three classes with molecules showing high (isoforms a, e,and j), intermediate (isoforms b, c, and f), and low/no IgE-binding activities (d, g, and l) [35].A study on the modulation of IgE reactivity by site-directed mutagenesis revealed a limitednumber of crucial amino acid positions (residues F30, S57, S112, I113, and D125 in the Bet v 1a se-quence) that strongly influence IgE binding [36]. Although Bet v 1 isoforms d, g, and l are high-ly similar in sequence to Bet v 1a (95.6%, 95.0%, 94.3% identity, respectively), thosehypoallergenic isoforms show variations in each of these positions. A small subset of criticalamino acids can drastically modulate the binding of IgE to an epitope and consequently changethe allergenicity of Bet v 1 isoforms as exemplified by Bet v 1 isoforms a and d [35,46]. In the
phosphate buffer, 50 mM NaCl at pH 7.0, and 10% 2H2O with Bruker Avance 700 MHz and Avance 800 MHzspectrometers. Chemical shift changes were mapped on Bet v 1a (pdb code: 1BV1, grey) or models of Bet v1d and Bet v 1m as in Fig 2F. Models of Bet v 1d and Bet v 1m were created using the Phyre server [92].Docked ligands [93] are illustrated in green sticks, oxygen in red. A Overlay of two 1H-15N HSQC spectra ofBet v 1a in the absence (black) and presence of a 15-fold excess of Q3OS (red). B Disappearing resonancesafter addition of Q3OSmapped on Bet v 1a in red. Q3OS is docked inside the hydrophobic pocket [17]. CMapping of chemical shift changes of (weak) Q3OClc or D (strong) Q3OGal interaction on Bet v 1a. EOverlay of two 1H-15N HSQC spectra of Bet v 1d in the absence (black) and presence of a 15-fold excess ofQ3OS (red) and F occurring chemical shift changes mapped on a model of Bet v 1d. Weak affinity isobserved for interaction of Bet v 1d with G Q3OGlc of H Q3OGal. I Overlay of two 1H-15N HSQC spectra ofBet v 1m in the absence (black) and presence of a 15-fold excess of Q3OS (red) and J occurring chemicalshift changes mapped on a model of Bet v 1m. High affinity is observed for the interaction of Bet v 1m with KQ3OGlc and L Q3OGal. Regions of the 1H-15N HSQC spectra during titration of Bet v 1d or Bet v 1m withQ3OGlc and Q3OGal are provided in the S1 Fig
doi:10.1371/journal.pone.0128677.g003
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 9 / 20
absence of ligands, we observed comparable IgE interactions (Fig 4A) and mediator release ac-tivities (Fig 4B) for isoforms a and m as measured by indirect ELISA and β-hexosaminidase re-lease from humanized rat basophil leukaemia (RBL) cells. Sequence and allergenicity of Bet v1m and the intermediate IgE-binding isoform Bet v 1b are nearly identical (Bet v 1.0201, 98.1%identity; [23]). The IgE-binding capacity of Bet v 1d is only marginal in the ELISA, and conse-quently an approximately 10-fold shift to a higher Bet v 1d concentration is needed for half-maximum release of β-hexosaminidase in comparison to the other isoforms (Fig 4A and 4B).Comparable results concerning the allergenicity of these Bet v 1 isoforms were also obtained inprevious experiment [35,36,46].
X-ray crystallography revealed that Bet v 1:ligand interaction could lead to an increase involume of the hydrophobic pocket, thus altering the protein surface [30,37], an effect that was
Fig 4. Interaction of Bet v 1 isoforms with serum IgE in the absence and presence of Q3OS. A, Binding of serial dilutions of pool serum IgE to equimolaramounts of surface-coated Bet v 1a, Bet v 1d, and Bet v 1m. Allergen-specific human IgE was detected with a horseradish peroxidase-conjugated mouseanti-human IgE antibody. As substrate 3,30,5,50-tetramethylbenzidine was used and the absorbance at 450 nm was measured after stopping the reaction with25% H2SO4. B, Mediator release induced by recombinant Bet v 1 isoforms. Humanized rat basophil leukemia cells were sensitized with a pool of humanbirch-specific sera. Cross-linking of membrane-bound human IgE by IgE-Bet v 1 isoform interaction and subsequent release of β-hexosaminidase wasdetermined with serial dilutions of Bet v 1 a, d and m. The β-hexosaminidase activity in the culture supernatants was quantified by photometricmeasurements. The percentage of β-hexosaminidase activity relative to cells lysed with Triton X-100 was calculated and corrected for spontaneous release.C, Binding of serial dilutions of pool serum IgE to equimolar amounts of surface-coated Bet v 1a, Bet v 1d, and Bet v 1m (as described in A) and D, mediatorrelease (as described in B) in the presence of a 5-molar excess of Q3OS.
doi:10.1371/journal.pone.0128677.g004
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 10 / 20
hypothesized to influence IgE epitopes. Our results, however, do not indicate any significant in-fluence of high-affinity ligands on the IgE binding properties of Bet v 1. Presence of a 5-foldmolar excess of Q3OS does not significantly influence the interaction of IgE with any of thethree isoforms (Fig 4C and 4D), and rutin, quercetin, Q3OGlc, Q3OGal, and sophorose didnot modify IgE-binding of the Bet v 1 isoforms either (S3 Fig). Our results are in agreementwith a recent study on the influence of deoxycholate on the allergenic properties of Bet v 1a[47].
Although recognition of an allergen by IgE is the key step in the allergic response, numerousother factors such as functional activity, presence of infective agents or chemical substancescan induce non-specific inflammatory responses or will augment the immunological shift to-wards an allergic reaction [48]. We suggest the lack of a direct ligand effect on IgE recognitionof Bet v 1, but leave open the possibility of indirect influences or sensitization [49]. Indeed, fla-vonoids influence the inflammatory pathway in human cells [50], and their uptake by thehuman body may be facilitated by Bet v 1 [51,52].
Bet v 1:flavonol-glycosides—adaptable sunscreens for birch pollenDNA?The Bet v 1:Q3OS complex was suggested to protect pollen DNA from UV-damage, and themixture of different isoforms was suggested to provide an individual fingerprint to preventself-pollination [17]. Indeed, glycosylated flavonoids are common in plant pollen. Flavonol-3-O-glycosides, e. g., were found in pollen from alder, ragweed, buttercup, date palm, narrow-leaf cattail, hazelnut, petunia, maize, and ophrys [11,53–60], and quercetin-3-O-glycosylgalac-toside was identified in pollen from Betula verrucosa [12] along with the Bet v 1a ligand Q3OS.Interactions of glycosylated flavonoids with different Bet v 1 isoforms in combination with var-iations in the production and composition of isoforms during maturation of pollen are proba-bly dependent on a set of parameters like climate, location, and solar radiation, as the Bet v 1levels in pollen are not constant over time [61], show variable IgE reactivity [27], and vary geo-graphically [26,62]. Upon UV-B radiation flavonoids (mostly quercetin derivatives) are pro-duced to protect the DNA from radiation damage [63] and glycosylation increases the UVtolerance of a flavonoid compared to the corresponding aglycon [64,65]. As we observed a shiftof the absorption maximum of quercetin depending on the sugar moiety (Fig 1B) and the ab-sorption maxima of different unglycosylated flavonoids shift towards higher (myricetin, quer-cetin, fisetin) or lower (naringenin) wavelengths during UV/VIS titration with Bet v 1 isoforms(S4 Table), Bet v 1 complex formation combined with variation of isoform composition in pol-len may be a means to expand or to optimize the absorption spectrum for sunlight-emittedUV-A radiation.
After maturation and before dispersing into the environment, the pollen dehydrate [66] toreduce their water content to 20% [67], thus forming highly viscous intracellular glass-likestructures [68]. In this milieu of highly concentrated biomolecules, glycosylated flavonoidsmay be protected from degradation or chemical modulation by complex formation with Bet v1.
Although flavonoids are considered most effective UV-B screening compounds because oftheir strong absorbance in the UV region [69], continuous UV-irradiation leads to their degra-dation [64]. Existence of functional complexes of glycosylated flavonoids and Bet v 1 in highconcentration may serve as an important signal for unharmed pollen DNA as UV-damage ofthe flavonoid moiety may modify the complex and prevent pollination. The pollen–pistil inter-action before fertilization comprises a series of complex cellular interactions involving a con-tinuous exchange of signals between pollen and the pistil of the stigma [70,71]. Upon contact,
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 11 / 20
birch pollen get rehydrated, and the Bet v 1-ligand complexes are released onto the stigma sur-face [10,66] with the specific mixture of the isoforms and ligands possibly serving as molecularfingerprints to prevent self-pollination.
This means that isoforms of PR-10-allergen do not simply just exist by chance, but havebeen selected through evolution with each isoform fulfilling a particular function. Isoformsfrom other Bet v 1 homologs like Ara h 8 [72,73], Dau c 1 [74,75], Api g 1 [76,77], Pru av 1[78,79] or Fra a 1 [80,81] seem to have less diverse functions in vivo without the necessity toprovide such a complex individual fingerprint. In those cases, the amount of (so far identified)genetically available and actually expressed isoforms seems to be significantly lower than ob-served for example for Mal d 1 in apple [82–87] or Bet v 1 in birch pollen [23,26,27].
Materials and Methods
FlavonoidsAll nonglycosylated and monoglycosylated flavonoids as well as glucose and galactose werepurchased in analytical grade from Sigma-Aldrich. Q3OS was obtained from ALNuMed (Ger-many) or AApin Chemicals Limited (UK).
Protein preparationThe genes coding for Bet v 1d (Bet v 1.0102; UniProt P43177) and Bet v 1m (Bet v 1.0204; Uni-Prot P43186) were purchased from GeneScript and cloned into the bacterial expression vectorpET11a (Novagen) using the restriction enzymes NdeI and BamHI-HF (New England Bio-labs). The expression for all isoforms was performed as previously described for Bet v 1a (Bet v1.0101, UniProt P15494, [17]) with minor modifications. For purification, Bet v 1 isoforms dand m were regained from protein pellets after cell lysis with 50 mM sodium phosphate, pH7.4, 200 mMNaCl, and 8 M urea and refolded by subsequently lowering the urea concentrationduring dialysis in 20 mMHepes buffer, pH 8.0 and 500 mMNaCl at 4°C (Bet v 1d) or 20 mMHepes buffer, pH 8.0 at RT (Bet v 1m).
Refolded Bet v 1d was further purified via hydrophobic interaction chromatography on a 4ml octyl sepharose column (HiTrap, Octyl Fast flow, GE Healthcare) equilibrated with loadingbuffer (20 mMHepes, pH 8.0, 1 M ammonium sulphate) and eluted stepwise with elution buff-er (20 mMHepes, pH 8.0). Refolded Bet v 1m was loaded on a 25 ml Q sepharose column (Qsepharose Fast flow, GE Healthcare) equilibrated with loading buffer (20 mMHepes, pH 8.0)followed by elution with 20 mMHepes, pH 8.0, 300 mMNaCl. Bet v 1a was purified as previ-ously described [17]. Fractions containing the respective Bet v 1 isoform were pooled and dia-lyzed at 4°C against 50 mM sodium phosphate, pH 7.0, 50 mM NaCl, concentrated and storedat -80°C. Protein concentrations were determined by the DC protein assay (BioRad) and UV/VIS spectroscopy using the molar extinction coefficient ε280 = 10430 M-1 cm-1. Standard meth-ods were used to analyse purity (SDS/PAGE), oligomeric state (size exclusion chromatogra-phy), and signal dispersion (1H-15N HSQC spectroscopy) of all isoforms (S4 Fig).
UV/VIS spectroscopyAll flavonoids and Bet v 1 isoforms were dissolved in 50 mM sodium phosphate, 50 mM NaCl,10% (v/v) DMSO, pH 7.0, to a final concentration of 10 to 20 μM in 500 μl. Absorption spectrafrom 200–800 nm were recorded at 25°C in a 1 cm quartz cuvette (Hellma GmbH) using a8453 UV-visible spectrophotometer (Agilent).
To observe binding of Q3OS to Bet v 1 isoforms a, d, and m, 20 μMQ3OS were initially in-cubated with 20 μM of the respective isoform in buffer without DMSO for 30 min at room
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 12 / 20
temperature (RT) in a total volume of 550 μl. Samples were concentrated to a final volume of100 μl using a Vivaspin concentrator (Sartorius, molecular mass cut off 10 kDa). The concen-trated samples where loaded on a G25 spin trap column (GE Healthcare) and eluted as de-scribed in the manual. Absorption spectra of the eluted fractions were normalized at 280 nmand set to zero at 700 nm.
To further characterize flavonoid binding to Bet v 1, titration experiments were performedby adding small amounts of concentrated Bet v 1 isoform a, d or m to different flavonoids.Changes of flavonoid absorption occurring at specific wavelengths were plotted against theprotein concentration. Prior to curve-fitting, absorbance data were corrected for dilution. Ifpossible, the equilibrium dissociation constant (Kd) was determined by non-linear regressionanalysis of the data with GraFit-5 (Version 5.0, Erithacus Software, UK) using the following Eq(1):
DA ¼ DAmax
2Q½ðBþ Qþ KdÞ�ððBþ Qþ KdÞ2�ð4BQÞ0:5Þ� ð1Þ
ΔAmax, maximum change in absorbance at specific wavelengths; B, Bet v 1a concentration; Q,total flavonoid concentration.
NMR spectroscopyAll NMR experiments were performed at 298 K in 50 mM sodium phosphate buffer, 50 mMNaCl, pH 7.0, 10% deuterium oxide (2H2O) with
15N-uniformly labelled Bet v 1 isoforms usingBruker Avance 700 MHz and Avance 800 MHz spectrometers with cryogenically cooled triple-resonance probes equipped with pulsed field-gradient capabilities. NMR data were processedusing NMRPipe [88] and visualized with NMRViewJ [89]. Three-dimensional 15N-editedNOESY (nuclear Overhauser enhancement spectroscopy, mixing times 120 ms) experimentsto assign chemical shifts were obtained with 500 μM 15N-labeled samples of Bet v 1 isoform dor m and yielded 91% of assigned residues for Bet v 1d and 89% for Bet v 1m. The sequence-specific assignments of the amide resonances of Bet v 1a are reported elsewhere [90].
Interaction of Q3OS with the Bet v 1 isoforms was measured by incubating 700 μMQ3OSwith 50 μM of each 15N-labeled Bet v 1 isoform in 50 mM sodium phosphate, 50 mM NaClbuffer, pH 7.0.
For titration experiments all other flavonoids were dissolved in deuterated DMSO, whileglucose and galactose were dissolved in 50 mM sodium phosphate buffer, 50 mM NaCl, pH7.0, 10% deuterium oxide and titrated stepwise to a final excess of up to 17-fold to protein sam-ples (ca. 100 μM). Final DMSO concentrations did not exceed 10% (v/v). Chemical shift per-turbations caused by increasing DMSO concentrations during measurements were identifiedby titrating DMSO in comparable steps. CSPs for ligand binding were calculated based on Eq(2):
ΔδHN and ΔδN, chemical shift differences of amide proton and nitrogen resonances, respective-ly, in ppm.
Kd values for flavonoid binding were determined with NMRViewJ [89]. All analysableamino acid residues that were unaffected by DMSO addition and showing CSPs> 0.08 ppmwere fitted to a quadratic binding curve with default settings, and an average Kd app was calcu-lated (Table 1 and S1–S3 Tables). The CSPs of all residues showing CSPs> 0.04 ppm were
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 13 / 20
mapped either on models of Bet v 1d and Bet v 1m or on the Bet v 1a structure (pdb code1BV1, [29]).
Sequence alignments, modelling and docking simulationSequence alignments of the Bet v 1 isoforms a, d and m were performed with ClustalW [91].Models of Bet v 1d and Bet v 1m were created using the Phyre2 server [92]. The calculatedmodels are based on the structural fold of PR-10 proteins with a confidence of 99% and a cov-erage of 92% (Bet v 1d) and 87% (Bet v 1m) compared to the template sequence. We usedAutoDockVina [93] to dock ligands into the hydrophobic pocket of Bet v 1a and the model ofBet v 1m. The PDB files for Q3OGlc and Q3OGal were created with ProDrg [94]. Furthermore,input files for Bet v 1a (pdb code 1BV1), the model of Bet v 1m, Q3OGlc, and Q3OGal weregenerated with AutoDockTools [95]. The grid box (2.0 nm×2.4 nm×2.8 nm, or 13.44 nm3) wascentred over the hydrophobic pocket of the isoforms and AutoDockVina was run with defaultsettings. Affinity scores were given by AutoDockVina as binding energies (ΔG), which weresubsequently used to calculate an equilibrium dissociation constant by Eq (3) withR = 0.001968 kcal�mol−1�K−1 and T = 298.15 K:
Kd ¼ e�DGRT ð3Þ
Ligand docking was performed only if more than five amino acids with Δδ� 0.12 ppm orintermediate exchange rates were observed during NMR titrations. The output of the dockingsimulation lists up to nine energetically most favourable orientations of the respective ligand inthe Bet v 1 pocket. The models in best agreement with our experimental NMR data were cho-sen to illustrate ligand binding to the Bet v 1 isoforms a or m.
Sera used in the studyFifteen sera of birch pollen-allergic subjects were collected, tested, and pooled according to theguideline of the European Medicines Agency (EMEA/CHMP/BWP/304831/2007). The serumpool is routinely used for batch-release testing of birch pollen-derived allergenic products atthe Paul-Ehrlich-Institut. The same serum pool was used for both, ELISA and mediatorrelease assays.
Indirect ELISA for IgE binding to Bet v 1 isoformsFor IgE-ELISA experiments, Maxisorp plates (Nunc via Fisher Scientific) were coated over-night at room temperature with 50 ng/100 μl recombinant Bet v 1 isoforms a, d, or m with a5-fold molar excess of quercetin-3-O-sophorose, rutin, quercetin, quercetin-3-O-glucoside,quercetin-3-O-galactoside, or sophorose, respectively, in phosphate-buffered saline (PBS).After blocking with PBS containing 2% bovine serum albumin (BSA) these plates and an un-coated control were incubated with a dilution series of a serum pool of birch-pollen allergicsubjects for 1 h at room temperature with PBS containing 0.05% Tween 20 and 0.1% BSA. Al-lergen-specific human IgE was detected with a horseradish peroxidase-conjugated mouse anti-human IgE antibody (Clone B3102E8, Southern biotech via Biozol, Eching, Germany) diluted1:1000. 3,30,5,50-tetramethylbenzidine (Roth, Karlsruhe) was used as substrate for horseradishperoxidase, and the absorbance at 450 nm was measured after stopping the reaction with 25%H2SO4.
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 14 / 20
β-Hexosaminidase release from humanized rat basophil leukemia (RBL)cellsThe mediator release assay was performed according to an established protocol [96]. Briefly,RBL cells expressing the α-chain of the high-affinity receptor for human IgE were sensitizedovernight at 37°C (5%CO2) with a serum pool of birch pollen-allergic subjects (diluted 1:40 inculture medium). After washing, cells were stimulated with serial dilutions of Bet v 1 isoformsa, d, or m in Tyrode's buffer containing 50% 2H2O. For complex formation, the Bet v 1 iso-forms were incubated overnight with a 5-fold molar excess of Q3OS, rutin, quercetin, Q3OGlc,Q3OGal, or sophorose, respectively, before stimulating the cells. Degranulation was quantifiedby photometric measurement of β-hexosaminidase activity in the culture supernatants. Thepercentage of β-hexosaminidase activity relative to cells lysed with Triton X-100 (Sigma-Al-drich, Steinheim, Germany) was calculated and corrected for spontaneous release (sensitizedcells without allergen).
Supporting InformationS1 Fig. Chemical structures of flavonoids used in this study. A flavone, B naringenin, C fise-tin,D quercetin, Emyricetin, F quercetin-3-O-glucoside, G quercetin-3-O-galactoside,Hquercetin-3-O-sophoroside.(TIF)
S2 Fig. NMR titration experiments of Q3OGlc and Q3OGal with the Bet v 1 isoforms. Theexperiments were performed with 100 μM 15N-uniformly labelled Bet v 1 isoforms at 298 K in50 mM sodium phosphate buffer, 50 mMNaCl at pH 7.0, and 10% 2H2O with Bruker Avance700 MHz and Avance 800 MHz spectrometers. Q3OGlc and Q3OGal were dissolved in deuter-ated DMSO and titrated stepwise to a final excess of up to 1:17 to protein samples. FinalDMSO concentrations did not exceed 10% (v/v). Spectra are illustrated in a divergent colourscheme from red (absence of ligand) to blue (final excess of ligand). Intermediate exchangingresidues are labelled. Titration experiments of Bet v 1a with AQ3OGlc and BQ3OGal, Bet v1d with CQ3OGlc,DQ3OGal and Bet v1m with EQ3OGlc and FQ3OGal.(TIF)
S3 Fig. Interaction of Bet v 1 isoforms with IgE in the presence of different flavonoids andsophorose. The left panel shows binding of serial dilutions of serum IgE to equimolar amountsof surface-coated Bet v 1a (■), Bet v 1d (●), and Bet v 1m (▲) with 5-molar excess of A querce-tin, C Q3OGlc, EQ3OGal, G sophorose, and I rutin respectively. Mediator release induced byrecombinant Bet v 1 isoforms is illustrated in the right panel. Humanized RBL cells were sensi-tized with a pool of human birch-specific sera. Cross-linking of membrane-bound human IgEby IgE-Bet v 1 isoform interaction and subsequent release of β-hexosaminidase was determinedwith serial dilutions of Bet v 1a (■), Bet v 1d (●), and Bet v 1m (▲) with 5-molar excess of Bquercetin,DQ3OGlc, F Q3OGal,H sophorose, and J rutin respectively.(TIF)
S4 Fig. Protein analytics. A SDS/PAGE on 19% gels of ca. 1 μg Bet v 1 isoforms (MW 17.4kDa) after purification. M, molecular-mass standard (Low Range, Bio-Rad Laboratories). BSEC of the isoforms performed with a Superdex S75 GL 10/300 column (total bed volume: 24ml; GE Healthcare) in 50 mM sodium phosphate, 50 mM NaCl, pH 7.0 at RT. Column calibra-tion was performed with conalbumin (75.0 kDa), ovalbumin (43.0 kDa), carbonic anhydrase(29.0 kDa) and ribonuclease (13.7 kDa). The elution profile of 0.5 mg Bet v 1a is shown inblack, 0.25 mg of Bet v 1d in red and 2.4 mg of Bet v 1m in blue. The peaks correspond to
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 15 / 20
monomeric proteins with molecular masses of 19.66 kDa (Bet v 1a), 17.63 kDa (Bet v 1d) and19.74 kDa (Bet v 1m). C 1H-15N HSQC spectra of 100 μM Bet v 1a (black), Bet v 1d (red) andBet v 1m (blue) in 50 mM sodium phosphate, 50 mMNaCl, pH 7.0 and 10% 2H2O at 298 K.(TIF)
S1 Table. Bet v 1a residues affected from addition of flavonoids with CSPs showingΔδnorm > 0.08 ppm.(DOCX)
S2 Table. Bet v 1m residues affected from addition of flavonoids with CSPs showingΔδnorm > 0.08 ppm.(DOCX)
S3 Table. Bet v 1d residues affected from addition of flavonoids with CSPs showing Δδnorm> 0.08 ppm.(DOCX)
S4 Table. Absorption maxima of unglycosylated flavonoids and their Bet v 1-complexes.(DOCX)
AcknowledgmentsWe thank Detlef Bartel and Frank Führer for providing the serum pool and Stefanie Randow,Ulrike Persau, and Ramona Heissmann for excellent technical assistance.
Author ContributionsConceived and designed the experiments: CSvL OHS DS RS SVMJH PR. Performed the exper-iments: CSvL TJ OHS LV CSG. Analyzed the data: CSvL TJ LV DS. Contributed reagents/ma-terials/analysis tools: DS CSG RS SV PR. Wrote the paper: CSvL DS MJH PR.
2. Ipsen H, Lowenstein H. Isolation and immunochemical characterization of the major allergen of birchpollen (Betula verrucosa). J Allergy Clin Immunol. 1983; 72: 150–159. PMID: 6886253
3. Vrtala S, Hirtenlehner K, Susani M, Akdis M, Kussebi F, Akdis CA, et al. Genetic engineering of a hypo-allergenic trimer of the major birch pollen allergen Bet v 1. FASEB J. 2001; 15: 2045–2047. PMID:11511511
4. Jarolim E, Rumpold H, Endler AT, Ebner H, Breitenbach M, Scheiner O, et al. IgE and IgG antibodies ofpatients with allergy to birch pollen as tools to define the allergen profile of Betula verrucosa. Allergy.1989; 44: 385–395. PMID: 2802112
5. Dreborg S. Food allergy in pollen-sensitive patients. Ann Allergy. 1988; 61: 41–46. PMID: 3061321
6. Geroldinger-Simic M, Zelniker T, Aberer W, Ebner C, Egger C, Greiderer A, et al. Birch pollen–relatedfood allergy: Clinical aspects and the role of allergen-specific IgE and IgG4 antibodies. J Allergy ClinImmunol. 2011; 127: 616–622.e1. doi: 10.1016/j.jaci.2010.10.027 PMID: 21251701
7. Liu J, Ekramoddoullah AKM. The family 10 of plant pathogenesis-related proteins: Their structure, regu-lation, and function in response to biotic and abiotic stresses. Physiol Mol Plant Pathol. 2006; 68: 3–13.
8. Casañal A, Zander U, Muñoz C, Dupeux F, Luque I, Botella MA, et al. The Strawberry Pathogenesis-re-lated 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding to Metabolic Intermediates.Journal of Biological Chemistry. 2013; 288: 35322–35332. doi: 10.1074/jbc.M113.501528 PMID:24133217
9. Bollen MA, Garcia A, Cordewener JH, Wichers HJ, Helsper JP, Savelkoul HF, et al. Purification andcharacterization of natural Bet v 1 from birch pollen and related allergens from carrot and celery. MolNutr Food Res. 2007; 51: 1527–1536. PMID: 17979095
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 16 / 20
10. Grote M, Vrtala S, Valenta R. Monitoring of two allergens, Bet v I and profilin, in dry and rehydratedbirch pollen by immunogold electron microscopy and immunoblotting. J Histochem Cytochem. 1993;41: 745–750. PMID: 8468456
12. Meurer B, Wiermann R, Strack D. Phenylpropanoid patterns in fagales pollen and their phylogenetic rel-evance. Phytochemistry. 1988; 27: 823–828.
13. Breiteneder H, Ferreira F, Hoffmann-Sommergruber K, Ebner C, Breitenbach M, Rumpold H, et al.Four recombinant isoforms of Cor a I, the major allergen of hazel pollen, show different IgE-bindingproperties. 1993; 212: 355–362. PMID: 7916686
14. Leja M, Kamińska I, Kramer M, Maksylewicz-Kaul A, Kammerer D, Carle R, et al. The Content of Phe-nolic Compounds and Radical Scavenging Activity Varies with Carrot Origin and Root Color. 2013; 68:163–170. doi: 10.1007/s11130-013-0351-3 PMID: 23613033
15. Hostetler GL, Riedl KM, Schwartz SJ. Effects of food formulation and thermal processing on flavones incelery and chamomile. Food Chem. 2013; 141: 1406–1411. doi: 10.1016/j.foodchem.2013.04.051PMID: 23790931
16. Koistinen KM, Soininen P, Venalainen TA, Hayrinen J, Laatikainen R, Perakyla M, et al. Birch PR-10cinteracts with several biologically important ligands. Phytochemistry. 2005; 66: 2524–2533. PMID:16246382
17. Seutter von Loetzen C, Hoffmann T, Hartl MJ, Schweimer K, SchwabW, Rösch P, et al. Secret of themajor birch pollen allergen Bet v 1: identification of the physiological ligand. Biochemical Journal. 2014;457: 379–390. doi: 10.1042/BJ20130413 PMID: 24171862
18. Mogensen JE, Wimmer R, Larsen JN, Spangfort MD, Otzen DE. The major birch allergen, Bet v 1,shows affinity for a broad spectrum of physiological ligands. J Biol Chem. 2002; 277: 23684–23692.PMID: 11953433
19. Hoffmann-Sommergruber K. Plant allergens and pathogenesis-related proteins. What do they have incommon? Int Arch Allergy Immunol. 2000; 122: 155–166. PMID: 10899758
20. Agarwal P, Agarwal P. Pathogenesis related-10 proteins are small, structurally similar but with diverserole in stress signaling. Mol Biol Rep. 2014; 41: 599–611. doi: 10.1007/s11033-013-2897-4 PMID:24343423
21. Lebel S, Schellenbaum P, Walter B, Maillot P. Characterisation of the Vitis vinifera PR10 multigenefamily. 2010; 10: 184. doi: 10.1186/1471-2229-10-184 PMID: 20727162
22. Breiteneder H, Pettenburger K, Bito A, Valenta R, Kraft D, Rumpold H, et al. The gene coding for themajor birch pollen allergen BetvI, is highly homologous to a pea disease resistance response gene.EMBO J. 1989; 8: 1935–1938. PMID: 2571499
23. Swoboda I, Jilek A, Ferreira F, Engel E, Hoffmann-Sommergruber K, Scheiner O, et al. Isoforms of Betv 1, the major birch pollen allergen, analyzed by liquid chromatography, mass spectrometry, and cDNAcloning. J Biol Chem. 1995; 270: 2607–2613. PMID: 7852325
24. Schenk M, Gilissen L, Esselink G, Smulders M. Seven different genes encode a diverse mixture of iso-forms of Bet v 1, the major birch pollen allergen. BMCGenomics. 2006; 7: 168. PMID: 16820045
25. Swoboda I, Scheiner O, Heberle-Bors E, Vicente O. cDNA cloning and characterization of three genesin the Bet v 1 gene family that encode pathogenesis-related proteins. 1995; 18: 865–874.
26. Erler A, Hawranek T, Krückemeier L, Asam C, Egger M, Ferreira F, et al. Proteomic profiling of birch(Betula verrucosa) pollen extracts from different origins. Proteomics. 2011; 11: 1486–1498. doi: 10.1002/pmic.201000624 PMID: 21360672
27. Schenk MF, Cordewener JH, America AH, Van't WestendeWP, Smulders MJ, Gilissen LJ. Characteri-zation of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen. BMCPlant Biol. 2009; 9: 24. doi: 10.1186/1471-2229-9-24 PMID: 19257882
28. Ferreira FD, Hoffmann-Sommergruber K, Breiteneder H, Pettenburger K, Ebner C, Sommergruber W,et al. Purification and characterization of recombinant Bet v I, the major birch pollen allergen. Immuno-logical equivalence to natural Bet v I. J Biol Chem. 1993; 268: 19574–19580. PMID: 8366100
29. Gajhede M, Osmark P, Poulsen FM, Ipsen H, Larsen JN, Joost van Neerven RJ, et al. X-ray and NMRstructure of Bet v 1, the origin of birch pollen allergy. Nat Struct Biol. 1996; 3: 1040–1045. PMID:8946858
30. Kofler S, Asam C, Eckhard U, Wallner M, Ferreira F, Brandstetter H. Crystallographically mapped li-gand binding differs in high and low IgE binding isoforms of birch pollen allergen bet v 1. J Mol Biol.2012; 422: 109–123. doi: 10.1016/j.jmb.2012.05.016 PMID: 22634284
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 17 / 20
31. Faber C, Lindemann A, Sticht H, Ejchart A, Kungl A, Susani M, et al. Secondary structure and tertiaryfold of the birch pollen allergen Bet v 1 in solution. J Biol Chem. 1996; 271: 19243–19250. PMID:8702605
32. Radauer C, Lackner P, Breiteneder H. The Bet v 1 fold: an ancient, versatile scaffold for binding oflarge, hydrophobic ligands. BMC Evol Biol. 2008; 8: 286. doi: 10.1186/1471-2148-8-286 PMID:18922149
33. Fernandes H, Michalska K, Sikorski M, Jaskolski M. Structural and functional aspects of PR-10 pro-teins. 2013; 280: 1169–1199. doi: 10.1111/febs.12114 PMID: 23289796
34. Roth-Walter F, Gomez-Casado C, Pacios LF, Mothes-Luksch N, Roth GA, Singer J, et al. Bet v 1 fromlipocalin-like protein acting as allergen only when devoid of iron by promoting Th2 lymphocytes. Journalof Biological Chemistry. 2014.
35. Ferreira F, Hirtenlehner K, Jilek A, Godnik-Cvar J, Breiteneder H, GrimmR, et al. Dissection of immuno-globulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potentialuse of hypoallergenic isoforms for immunotherapy. J Exp Med. 1996; 183: 599–609. PMID: 8627171
36. Ferreira F, Ebner C, Kramer B, Casari G, Briza P, Kungl AJ, et al. Modulation of IgE reactivity of aller-gens by site-directed mutagenesis: potential use of hypoallergenic variants for immunotherapy. FASEBJ. 1998; 12: 231–242. PMID: 9472988
37. Markovic-Housley Z, Degano M, Lamba D, von Roepenack-Lahaye E, Clemens S, Susani M, et al.Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely bio-logical function as a plant steroid carrier. J Mol Biol. 2003; 325: 123–133. PMID: 12473456
38. Göbl C, Madl T, Simon B, Sattler M. NMR approaches for structural analysis of multidomain proteinsand complexes in solution. Prog Nucl Magn Reson Spectrosc. 2014; 80: 26–63. doi: 10.1016/j.pnmrs.2014.05.003 PMID: 24924266
40. Srinivas K, King JW, Howard LR, Monrad JK. Solubility and solution thermodynamic properties of quer-cetin and quercetin dihydrate in subcritical water. J Food Eng. 2010; 100: 208–218.
41. TaoW, Yang N, Duan JA, Wu D, Guo J, Tang Y, et al. Simultaneous determination of eleven major fla-vonoids in the pollen of Typha angustifolia by HPLC-PDA-MS. Phytochem Anal. 2011; 22: 455–461.doi: 10.1002/pca.1302 PMID: 22033915
42. Holgersson J, Gustafsson A, Breimer ME. Characteristics of protein-carbohydrate interactions as abasis for developing novel carbohydrate-based antirejection therapies. Immunol Cell Biol. 2005; 83:694–708. PMID: 16266322
43. Toone EJ. Structure and energetics of protein-carbohydrate complexes. Curr Opin Struct Biol. 1994; 4:719–728.
44. Lemieux RU. HowWater Provides the Impetus for Molecular Recognition in Aqueous Solution. AccChem Res. 1999; 32: 631–631.
45. Swaminathan CP, Surolia N, Surolia A. Role of Water in the Specific Binding of Mannose and Mannooli-gosaccharides to Concanavalin A. J Am Chem Soc. 1998; 120: 5153–5159.
46. Arquint O, Helbling A, Crameri R, Ferreira F, Breitenbach M, Pichler WJ. Reduced in vivo allergenicityof Bet v 1d isoform, a natural component of birch pollen. J Allergy Clin Immunol. 1999; 104: 1239–1243.PMID: 10589007
47. Asam C, Batista AL, Moraes AH, de Paula VS, Almeida FC, Aglas L, et al. Bet v 1—a Trojan horse forsmall ligands boosting allergic sensitization? Clin Exp Allergy. 2014.
48. Bufe A. The biological function of allergens: relevant for the induction of allergic diseases? Int Arch Al-lergy Immunol. 1998; 117: 215–219. PMID: 9876222
49. Bublin M, Eiwegger T, Breiteneder H. Do lipids influence the allergic sensitization process? J AllergyClin Immunol. 2014.
50. Chirumbolo S. The role of quercetin, flavonols and flavones in modulating inflammatory cell function. In-flammation & Allergy-Drug Targets. 2010; 9: 263–285.
51. Mogensen JE, Ferreras M, Wimmer R, Petersen SV, Enghild JJ, Otzen DE. The Major Allergen fromBirch Tree Pollen, Bet v 1, Binds and Permeabilizes Membranes. Biochemistry. 2007; 46: 3356–3365.PMID: 17311414
52. Golebski K, Roschmann KI, Toppila-Salmi S, Hammad H, Lambrecht BN, Renkonen R, et al. The multi-faceted role of allergen exposure to the local airway mucosa. Allergy. 2013; 68: 152–160. doi: 10.1111/all.12080 PMID: 23240614
53. Karioti A, Kitsaki CK, Zygouraki S, Ziobora M, Djeddi S, Skaltsa H, et al. Occurrence of flavonoids inOphrys (Orchidaceae) flower parts. 2008; 203: 602–609.
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 18 / 20
54. Mo Y, Nagel C, Taylor LP. Biochemical complementation of chalcone synthase mutants defines a rolefor flavonols in functional pollen. Proc Natl Acad Sci U S A. 1992; 89: 7213–7217. PMID: 11607312
55. Ceska O, Styles ED. Flavonoids from Zea mays pollen. Phytochemistry. 1984; 23: 1822–1823.
56. STEVENS FA, MOORE D, BAER H. The isolation of isoquercitrin from giant ragweed pollen; the elec-trophoretic pattern and biologic activity of the pigment. J Allergy. 1951; 22: 165–169. PMID: 14823829
57. Sosa F, Percheron F. Isolation and identification of a quercetol-3-sophoroside in Alnus cordata Desf.pollen. C R Acad Sci Hebd Seances Acad Sci D. 1965; 261: 4544–4546. PMID: 4954804
58. EL RIDI MS, STRAIT LA, ABOULWAFAMH. Isolation of rutin from the pollen grain of the date palm(Dactylifera Palma L.). Arch Biochem Biophys. 1952; 39: 317–321. PMID: 12997166
59. Jia SS, Liu YL, Ma CM, Yang SL, Zhou HM, Zhao DC, et al. Studies on the constituents of the flavo-noids from the pollen of Typha angustfolia L. (puhuang). Yao Xue Xue Bao. 1986; 21: 441–446. PMID:3811929
60. Arraez-Roman D, Zurek G, Bässmann C, Almaraz-Abarca N, Quirantes R, Segura-Carretero A, et al.Identification of phenolic compounds from pollen extracts using capillary electrophoresis-electrospraytime-of-flight mass spectrometry. Anal Bioanal Chem. 2007; 389: 1909–1917. PMID: 17899027
61. Buters JTM, Weichenmeier I, Ochs S, Pusch G, KreylingW, Boere AJF, et al. The allergen Bet v 1 infractions of ambient air deviates from birch pollen counts. Allergy. 2010; 65: 850–858. doi: 10.1111/j.1398-9995.2009.02286.x PMID: 20132158
62. Buters JTM, Kasche A, Weichenmeier I, Schober W, Klaus S, Traidl-Hoffmann C, et al. Year-to-YearVariation in Release of Bet v 1 Allergen from Birch Pollen: Evidence for Geographical Differences be-tweenWest and South Germany. Int Arch Allergy Immunol. 2008; 145: 122–130. PMID: 17848805
63. Lavola A, Nybakken L, Rousi M, Pusenius J, Petrelius M, Kellomäki S, et al. Combination treatment ofelevated UVB radiation, CO2 and temperature has little effect on silver birch (Betula pendula) growthand phytochemistry. Physiol Plantarum. 2013; 149: 499–514.
64. Cvetković D, Markovic D, Cvetković D, Radovanovic B. Effects of continuous UV-irradiation on the anti-oxidant activities of quercetin and rutin in solution in the presence of lecithin as the protective target. JSerb Chem Soc. 2011; 76: 973–985.
65. Smith GJ, Thomsen SJ, Markham KR, Andary C, Cardon D. The photostabilities of naturally occurring5-hydroxyflavones, flavonols, their glycosides and their aluminium complexes. J Photochem PhotobiolA. 2000; 136: 87–91.
66. Firon N, Nepi M, Pacini E. Water status and associated processes mark critical stages in pollen devel-opment and functioning. Annals of Botany. 2012; 109: 1201–1214. doi: 10.1093/aob/mcs070 PMID:22523424
67. Pacini E. Cell biology of anther and pollen development. In: Williams E, Clarke A, Knox RB, editors.:Springer Netherlands; 1994. pp. 289–308.
68. Buitink J, Claessens MMAE, Hemminga MA, Hoekstra FA. Influence of Water Content and Tempera-ture on Molecular Mobility and Intracellular Glasses in Seeds and Pollen. Plant Physiol. 1998; 118:531–541. PMID: 9765538
69. Morales LO, Tegelberg R, Brosché M, Lindfors A, Siipola S, Aphalo PJ. Temporal variation in epidermalflavonoids due to altered solar UV radiation is moderated by the leaf position in Betula pendula. PhysiolPlantarum. 2011; 143: 261–270. doi: 10.1111/j.1399-3054.2011.01511.x PMID: 21883252
70. Heslop-Harrison J. Incompatibility and the Pollen-Stigma Interaction. Annu Rev Plant Physiol. 1975;26: 403–425.
71. Hiscock SJ, Allen AM. Diverse cell signalling pathways regulate pollen-stigma interactions: the searchfor consensus. New Phytol. 2008; 179: 286–317. doi: 10.1111/j.1469-8137.2008.02457.x PMID:19086285
72. Mittag D, Akkerdaas J, Ballmer-Weber B, Vogel L, Wensing M, Becker W, et al. Ara h 8, a Bet v 1–-homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanutallergy. J Allergy Clin Immunol. 2004; 114: 1410–1417. PMID: 15577846
73. Riecken S, Lindner B, Petersen A, Jappe U, Becker WM. Purification and characterization of naturalAra h 8, the Bet v 1 homologous allergen from peanut, provides a novel isoform. Biol Chem. 2008; 389:415–423. doi: 10.1515/BC.2008.038 PMID: 18208358
74. Yamamoto M, Torikai S, Oeda K. Amajor root protein of carrots with high homology to intracellular path-ogenesis-related (PR) proteins and pollen allergens. Plant Cell Physiol. 1997; 38: 1080–1086. PMID:9360325
75. Ballmer-Weber BK, Wangorsch A, Bohle B, Kaul S, Kündig T, Fötisch K, et al. Component-resolved invitro diagnosis in carrot allergy: Does the use of recombinant carrot allergens improve the reliability ofthe diagnostic procedure?. 2005; 35: 970–978. PMID: 16008686
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 19 / 20
76. Breiteneder H, Hoffmann-Sommergruber K, O'Riordain G, Susani M, Ahorn H, Ebner C, et al. Molecularcharacterization of Api g 1, the major allergen of celery (Apium graveolens), and its immunological andstructural relationships to a group of 17-kDa tree pollen allergens. Eur J Biochem. 1995; 233: 484–489.PMID: 7588792
77. Hoffmann-Sommergruber K, Ferris R, Pec M, Radauer C, O’Riordain G, Laimer da CM, et al. Charac-terization of Api g 1.0201, a NewMember of the Api g 1 Family of Celery Allergens. Int Arch AllergyImmunol. 2000; 122: 115–123. PMID: 10878490
78. Scheurer S, Metzner K, Haustein D, Vieths S. Molecular cloning, expression and characterization ofPru a 1, the major cherry allergen. Mol Immunol. 1997; 34: 619–629. PMID: 9393965
79. Reuter A, Fortunato D, Garoffo LP, Napolitano L, Scheurer S, Giuffrida MG, et al. Novel isoforms of Pruav 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of mo-lecular biology and proteomics. Proteomics. 2005; 5: 282–289. PMID: 15593144
80. Karlsson AL, Alm R, Ekstrand B, Fjelkner-Modig S, Schiott A, Bengtsson U, et al. Bet v 1 homologuesin strawberry identified as IgE-binding proteins and presumptive allergens. Allergy. 2004; 59: 1277–1284. PMID: 15507096
81. Muñoz C, Hoffmann T, Escobar NM, Ludemann F, Botella MA, Valpuesta V, et al. The strawberry fruitFra a allergen functions in flavonoid biosynthesis. Mol Plant. 2010; 3: 113–124. doi: 10.1093/mp/ssp087 PMID: 19969523
82. Vanekkrebitz M, Hoffmannsommergruber K, Machado MLD, Susani M, Ebner C, Kraft D, et al. Cloningand Sequencing of Mal d 1, the Major Allergen from Apple (Malus domestica), and Its ImmunologicalRelationship to Bet v 1, the Major Birch Pollen Allergen. Biochem Biophys Res Commun. 1995; 214:538–551. PMID: 7677763
83. Ziadi S, Poupard P, Brisset M, Paulin J, Simoneau P. Characterization in apple leaves of two sub-classes of PR-10 transcripts inducible by acibenzolar-S-methyl, a functional analogue of salicylic acid.Physiol Mol Plant Pathol. 2001; 59: 33–43.
84. Atkinson RG, Perry J, Matsui T, Ross GS, Macrae EA. A stress-, pathogenesis-, and allergen-relatedcDNA in apple fruit is also ripening-related. N Z J Crop Hortic Sci. 1996; 24: 103–107.
85. Pühringer H, Moll D, Hoffmann-Sommergruber K, Watillon B, Katinger H, da Câmara Machado, MargitLaimer. The promoter of an apple Ypr10 gene, encoding the major allergen Mal d 1, is stress- and path-ogen-inducible. 2000; 152: 35–50.
86. Holm J, Baerentzen G, Gajhede M, Ipsen H, Larsen JN, Lowenstein H, et al. Molecular basis of allergiccross-reactivity between group 1 major allergens from birch and apple. J Chromatogr B Biomed SciAppl. 2001; 756: 307–313. PMID: 11419722
87. Hoffmann-Sommergruber K, Vanek-Krebitz M, Radauer C, Wen J, Ferreira F, Scheiner O, et al. Geno-mic characterization of members of the Bet v 1 family: genes coding for allergens and pathogenesis-re-lated proteins share intron positions. Gene. 1997; 197: 91–100. PMID: 9332353
88. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A. NMRPipe: a multidimensional spectral pro-cessing system based on UNIX pipes. J Biomol NMR. 1995; 6: 277–293. PMID: 8520220
89. Johnson BA, Blevins RA. NMRview: A computer program for the visualization and analysis of NMRdata. J Biomol NMR. 1994; 4: 603–614. doi: 10.1007/BF00404272 PMID: 22911360
90. Schweimer K, Sticht H, Nerkamp J, BoehmM, Breitenbach M, Vieths S, et al. NMR Spectroscopy Re-veals Common Structural Features of the Birch Pollen Allergen Bet v 1 and the Cherry Allergen Pru a 1.Appl Magn Reson. 1999; 17: 449–456.
91. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W andClustal X version 2.0. Bioinformatics. 2007; 23: 2947–2948. PMID: 17846036
92. Kelley LA, Sternberg MJ. Protein structure prediction on theWeb: a case study using the Phyre server.Nat Protoc. 2009; 4: 363–371. doi: 10.1038/nprot.2009.2 PMID: 19247286
93. Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoringfunction, efficient optimization, and multithreading. J Comput Chem. 2010; 31: 455–461. doi: 10.1002/jcc.21334 PMID: 19499576
94. Schüttelkopf AW, van Aalten DM. PRODRG: a tool for high-throughput crystallography of protein-ligandcomplexes. Acta Crystallogr D Biol Crystallogr. 2004; 60: 1355–1363. PMID: 15272157
96. Vogel L, Lüttkopf D, Hatahet L, Haustein D, Vieths S. Development of a functional in vitro assay as anovel tool for the standardization of allergen extracts in the human system. Allergy. 2005; 60: 1021–1028. PMID: 15969682
Bet v 1 and Ligand Recognition
PLOS ONE | DOI:10.1371/journal.pone.0128677 June 4, 2015 20 / 20
